The evolution of antimicrobial peptide resistance in Pseudomonas aeruginosa is shaped by strong epistatic interactions.

Colistin is an antimicrobial peptide that has become the only remaining alternative for the treatment of multidrug-resistant Gram-negative bacterial infections, but little is known of how clinical levels of colistin resistance evolve. We use in vitro experimental evolution and whole-genome sequencing of colistin-resistant Pseudomonas aeruginosa isolates from cystic fibrosis patients to reconstruct the molecular evolutionary pathways open for high-level colistin resistance. We show that the evolution of resistance is a complex, multistep process that requires mutation in at least five independent loci that synergistically create the phenotype. Strong intergenic epistasis limits the number of possible evolutionary pathways to resistance. Mutations in transcriptional regulators are essential for resistance evolution and function as nodes that potentiate further evolution towards higher resistance by functionalizing and increasing the effect of the other mutations. These results add to our understanding of clinical antimicrobial peptide resistance and the prediction of resistance evolution.

Within-host evolution of Pseudomonas aeruginosa reveals adaptation toward iron acquisition from hemoglobin.

ABSTRACT Pseudomonas aeruginosa airway infections are a major cause of mortality and morbidity of cystic fibrosis (CF) patients. In order to persist, P. aeruginosa depends on acquiring iron from its host, and multiple different iron acquisition systems may be active during infection. This includes the pyoverdine siderophore and the Pseudomonas heme utilization (phu) system. While the regulation and mechanisms of several iron-scavenging systems are well described, it is not clear whether such systems are targets for selection during adaptation of P. aeruginosa to the host environment. Here we investigated the within-host evolution of the transmissible P. aeruginosa DK2 lineage. We found positive selection for promoter mutations leading to increased expression of the phu system. By mimicking conditions of the CF airways in vitro, we experimentally demonstrate that increased expression of phuR confers a growth advantage in the presence of hemoglobin, thus suggesting that P. aeruginosa evolves toward iron acquisition from hemoglobin. To rule out that this adaptive trait is specific to the DK2 lineage, we inspected the genomes of additional P. aeruginosa lineages isolated from CF airways and found similar adaptive evolution in two distinct lineages (DK1 and PA clone C). Furthermore, in all three lineages, phuR promoter mutations coincided with the loss of pyoverdine production, suggesting that within-host adaptation toward heme utilization is triggered by the loss of pyoverdine production. Targeting heme utilization might therefore be a promising strategy for the treatment of P. aeruginosa infections in CF patients. IMPORTANCE Most bacterial pathogens depend on scavenging iron within their hosts, which makes the battle for iron between pathogens and hosts a hallmark of infection. Accordingly, the ability of the opportunistic pathogen Pseudomonas aeruginosa to cause chronic infections in cystic fibrosis (CF) patients also depends on iron-scavenging systems. While the regulation and mechanisms of several such iron-scavenging systems have been well described, not much is known about how the within-host selection pressures act on the pathogens' ability to acquire iron. Here, we investigated the within-host evolution of P. aeruginosa, and we found evidence that P. aeruginosa during long-term infections evolves toward iron acquisition from hemoglobin. This adaptive strategy might be due to a selective loss of other iron-scavenging mechanisms and/or an increase in the availability of hemoglobin at the site of infection. This information is relevant to the design of novel CF therapeutics and the development of models of chronic CF infections.

Archetypal analysis of diverse Pseudomonas aeruginosa transcriptomes reveals adaptation in cystic fibrosis airways.

BACKGROUND: Analysis of global gene expression by DNA microarrays is widely used in experimental molecular biology. However, the complexity of such high-dimensional data sets makes it difficult to fully understand the underlying biological features present in the data.The aim of this study is to introduce a method for DNA microarray analysis that provides an intuitive interpretation of data through dimension reduction and pattern recognition. We present the first "Archetypal Analysis" of global gene expression. The analysis is based on microarray data from five integrated studies of Pseudomonas aeruginosa isolated from the airways of cystic fibrosis patients. RESULTS: Our analysis clustered samples into distinct groups with comprehensible characteristics since the archetypes representing the individual groups are closely related to samples present in the data set. Significant changes in gene expression between different groups identified adaptive changes of the bacteria residing in the cystic fibrosis lung. The analysis suggests a similar gene expression pattern between isolates with a high mutation rate (hypermutators) despite accumulation of different mutations for these isolates. This suggests positive selection in the cystic fibrosis lung environment, and changes in gene expression for these isolates are therefore most likely related to adaptation of the bacteria. CONCLUSIONS: Archetypal analysis succeeded in identifying adaptive changes of P. aeruginosa. The combination of clustering and matrix factorization made it possible to reveal minor similarities among different groups of data, which other analytical methods failed to identify. We suggest that this analysis could be used to supplement current methods used to analyze DNA microarray data.

Evolutionary remodeling of global regulatory networks during long-term bacterial adaptation to human hosts.

The genetic basis of bacterial adaptation to a natural environment has been investigated in a highly successful Pseudomonas aeruginosa lineage (DK2) that evolved within the airways of patients with cystic fibrosis (CF) for more than 35 y. During evolution in the CF airways, the DK2 lineage underwent substantial phenotypic changes, which correlated with temporal fixation of specific mutations in the genes mucA (frame-shift), algT (substitution), rpoN (substitution), lasR (deletion), and rpoD (in-frame deletion), all encoding regulators of large gene networks. To clarify the consequences of these genetic changes, we moved the specific mutations, alone and in combination, to the genome of the reference strain PAO1. The phenotypes of the engineered PAO1 derivatives showed striking similarities with phenotypes observed among the DK2 isolates. The phenotypes observed in the DK2 isolates and PAO1 mutants were the results of individual, additive and epistatic effects of the regulatory mutations. The mutations fixed in the σ factor encoding genes algT, rpoN, and rpoD caused minor changes in σ factor activity, resulting in remodeling of the regulatory networks to facilitate generation of unexpected phenotypes. Our results suggest that adaptation to a highly selective environment, such as the CF airways, is a highly dynamic and complex process, which involves continuous optimization of existing regulatory networks to match the fluctuations in the environment.

Mutations in 23S rRNA confer resistance against azithromycin in Pseudomonas aeruginosa.

The emergence of antibiotic-resistant Pseudomonas aeruginosa is an important concern in the treatment of long-term airway infections in cystic fibrosis patients. In this study, we report the occurrence of azithromycin resistance among clinical P. aeruginosa DK2 isolates. We demonstrate that resistance is associated with specific mutations (A2058G, A2059G, and C2611T in Escherichia coli numbering) in domain V of 23S rRNA and that introduction of A2058G and C2611T into strain PAO1 results in azithromycin resistance.

Polysaccharides serve as scaffold of biofilms formed by mucoid Pseudomonas aeruginosa.

Chronic lung infection by mucoid Pseudomonas aeruginosa is one of the major pathologic features in patients with cystic fibrosis. Mucoid P. aeruginosa is notorious for its biofilm forming capability and resistance to immune attacks. In this study, the roles of extracellular polymeric substances from biofilms formed by mucoid P. aeruginosa were investigated. Alginate is not an essential structure component for mucoid P. aeruginosa biofilms. Genetic studies revealed that Pel and Psl polysaccharides serve as essential scaffold and mediate macrocolony formation in mucoid P. aeruginosa biofilms. The Psl polysaccharide is more important than Pel polysaccharide in mucoid P. aeruginosa biofilm structure maintenance and phagocytosis resistance. The polysaccharides were further found to protect mucoid P. aeruginosa strain from host immune clearance in a mouse model of acute lung infection.

Evolutionary dynamics of bacteria in a human host environment.

Laboratory evolution experiments have led to important findings relating organism adaptation and genomic evolution. However, continuous monitoring of long-term evolution has been lacking for natural systems, limiting our understanding of these processes in situ. Here we characterize the evolutionary dynamics of a lineage of a clinically important opportunistic bacterial pathogen, Pseudomonas aeruginosa, as it adapts to the airways of several individual cystic fibrosis patients over 200,000 bacterial generations, and provide estimates of mutation rates of bacteria in a natural environment. In contrast to predictions based on in vitro evolution experiments, we document limited diversification of the evolving lineage despite a highly structured and complex host environment. Notably, the lineage went through an initial period of rapid adaptation caused by a small number of mutations with pleiotropic effects, followed by a period of genetic drift with limited phenotypic change and a genomic signature of negative selection, suggesting that the evolving lineage has reached a major adaptive peak in the fitness landscape. This contrasts with previous findings of continued positive selection from long-term in vitro evolution experiments. The evolved phenotype of the infecting bacteria further suggests that the opportunistic pathogen has transitioned to become a primary pathogen for cystic fibrosis patients.